Potassium Channels and Dendritic Function in Hippocampal Pyramidal Neurons

海马锥体神经元的钾通道和树突功能

• 批准号: 7594222
• 负责人: Dax A Hoffman
• 金额: $ 75.43万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594222
• 关键词: Action Potentials; Acute; Affect; Alzheimer' s Disease; Architecture; Axon; Back; Biophysics; Brain; Brain region; Cell physiology; Cells; Chemosensitization; Chromosome Pairing; Collaborations; Complement; Computer Simulation; Condition; Data; Dendrites; Dendritic Spines; Dominant-Negative Mutation; Doxycycline; Emotions; Epilepsy; Event; Excision; Exhibits; Family; Fire - disasters; Fluorescence; Frequencies; Gene Expression; Gene Proteins; Genes; Green Fluorescent Proteins; Head; Hippocampus (Brain); Image; Imaging Techniques; Kinetics; Knowledge; Kv4.2 channel; Label; Learning; Life; Maintenance; Measures; Memory; Modification; Molecular; Molecular Cloning; Morphologic artifacts; Mus; Mutation; NR1 gene; Nature; Neuraxis; Neurons; Numbers; Pattern; Phase; Physiological; Potassium Channel; Predisposition; Property; Protein Overexpression; Proteins; Protocols documentation; Recovery; Resistance; Role; Second Messenger Systems; Seizures; Shapes; Signal Transduction; Sindbis Virus; Slice; Synapses; Synaptic plasticity; Synaptophysin; System; Techniques; Tetracycline; Tetracyclines; Time; Trans-Activators; Transgenic Mice; United States National Institutes of Health; Variant; Vertebral column; Virus; Voltage-Gated Potassium Channel; Width; Work; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; amino 3 hydroxy 5 methylisoxazole 4 propionate; density; dentate gyrus; electrical property; experience; hippocampal pyramidal neuron; human NR1 protein; morris water maze; mutant; neuronal cell body; neurophysiology; patch clamp; promoter; research study; response; second messenger; size; trafficking; voltage; voltage clamp; voltage gated channel

In the dendrites of hippocampal CA1 pyramidal neurons, a nonuniform density of subthreshold, rapidly inactivating potassium channels regulates signal propagation. This nonuniform distribution (with higher expression in the dendrites than in the soma) means that the electrical properties of the dendrites are markedly different from those of the soma. Incoming synaptic signals are shaped by the activity of these channels, and action potentials, once initiated in the axon, progressively decrease in amplitude as they propagate back into the dendrites. Combining patch clamp recording with molecular techniques, the Molecular Neurophysiology and Biophysics Unit investigates the electrophysiological properties and molecular nature of the voltage-gated channels expressed in CA1 dendrites, how their expression is regulated, and what their role is in learning and memory. Kv4.2 control of firing patterns in hippocampal CA1 pyramidal neurons. Although recent molecular cloning studies have found several families of voltage-gated K channel genes expressed in the mammalian brain, at present, information regarding the relationship between the protein products of these genes and their various neuronal functions is lacking. Our lab has used a combination of molecular, electrophysiological, imaging techniques to show that the voltage gated potassium channel subunit Kv4.2 controls AP half-width, frequency-dependent AP broadening and dendritic action potential propagation. As Ca2 influx occurs primarily during AP repolarization, Kv4.2 activity can regulate cellular processes involving Ca2-dependent second messenger cascades such as gene expression and synaptic plasticity. We are currently developing techniques which will enable us to directly record from dendrites where we have altered voltage-gated channel functional expression. Kv4.2 trafficking in CA1 pyramidal neuron dendrites. Using a modified Sinbis virus system to overexpress EGFP-labeled Kv4.2 (Kv4.2g) in cultured hippocampal neurons, we found that the EGFP fluorescence in dendritic spines of Kv4.2g expressing neurons appeared brighter than that from the adjacent dendritic shaft. The ratio of spine head to dendritic shaft fluorescence in Kv4.2g expressing neurons was approximately two-fold greater than in neurons expressing EGFP. Kv4.2 expression in spines was further shown using electronmicroscopy in collaboration with Ron Petralia here at the NIH. We found stimulation (AMPA) to result in an activity-dependent redistribution of Kv4.2g away from spines to the dendritic shaft and a punctate accumulation of Kv4.2g within the soma. This AMPA-induced redistribution of Kv4.2g occurred within 15 min of stimulation and was reversible, indicating that the treatment was not excitotoxic (6 h washout). Co-expression with pre- and post-synaptic markers (synaptophysin and NR1) showed that Kv4.2 undergoes activity-induced redistribution without a gross change in synaptic architecture or number. We have confirmed these findings with live imaging of Kv4.2g removal from the spine in response to AMPA stimulation. The large number of synapses stimulated in these conditions enabled us to directly measure the effect of internalization as a decrease in the endogenous whole-cell transient K current from uninfected hippocampal neurons without a change in sustained or non-inactivating delayed rectifier-type voltage-gated K current amplitudes. Thus, activity-dependent Kv4.2 internalization occurs natively and is not an artifact of overexpression. We are currently investigating the requirements and mechanisms of Kv4.2 activity-dependent trafficking. Role of voltage-gated potassium channels in synaptic plasticity. Using the Sindbis virus system to infect organotypic slice cultures with Kv4.2g and Kv4.2g(W362F), we have begun investigating the role of Kv4.2 in LTP using a depolarization pairing protocol. For the first 10 min after pairing, potentiation is similar in all three groups, achieving 100% increase in EPSC size. After this period, however, Kv4.2 overexpressing neurons fail to maintain potentiation such that EPSC size is back to baseline after 25 min. Conversely, expression of Kv4.2g(W362F) results in a potentiation, which reaches a greater level 40-50 min after initiation, compared to controls. These data indicate that Kv4.2 channels modulate the degree of LTP by influencing the induction of a late phase of potentiation or by controlling the mechanisms of LTP maintenance. We are currently characterizing the mechanisms of Kv4.2s effect on LTP. Creation and characterization of Kv4.2 transgenic mice. We are currently characterizing a transgenic mouse expressing a dominant negative pore mutation in the voltage-gated potassium channel subunit Kv4.2. This mouse expresses the mutant Kv4.2 channel along with GFP under control of a tetracycline transactivator (tTA) responsive promoter. Expression is spatially controlled by a new line of tTA expressing mice that limit tTA activity to the CA1 and dentate gyrus regions of the hippocampus. Expression can be controlled temporally by administration of doxycycline. Experiments in acute hippocampal slices from these mice will be used to investigate Kv4.2s role in regulating AP backpropagation into CA1 dendrites and in synaptic integration and plasticity. In collaboration with Dr. Anne Anderson, we are investigating seizure susceptibility in these mice. In addition, we are using these mice to investigate Kv4.2s role in hippocampal dependent learning and memory in the Morris water maze. Role of auxiliary proteins in regulating Kv4.2 expression and function. A-type K currents have unique kinetic and voltage-dependent properties that allow them to finely tune synaptic events, action potentials and neuronal firing. To achieve this diversity, different neuron types express specific complements of Kv4.2 auxiliary subunits. In hippocampal CA1 pyramidal neurons, DPPX is a prominently expressed subunit, which restores many properties of native CA1 A-type currents when co-expressed with Kv4.2 in heterologous systems. To investigate the physiological role of DPPX in CA1 neurons we developed, in collaboration with Bernardo Rudys lab, short-interfering RNAs (siRNAs) to suppress the expression of all DPPX variants. To investigate whether DPPX alters the kinetics of A-type currents in a native system, we conducted voltage-clamp experiments in outside-out patches from CA1 pyramidal neurons in hippocampal organotypic slices infected with siDPPX using a modified Sindbis virus system. We found that siDPPX results in a delayed recovery from inactivation and rightward shifted the steady-state inactivation and activation curves for A-type currents. To determine the physiological effect of the A-type current kinetic modifications by siDPPX, we carried out current-clamp experiments in siDPPX expressing cells. Compared to negative control, siDPPX-infected neurons exhibited decreased input resistance, delayed time to AP onset, increased AP threshold, increased AP half-width and reduced fast AHP amplitudes. Thus siDPPX had contrasting effects, decreasing excitability subthreshold and increasing excitability suprathreshold. We have used computer modeling to determine which of these sub- and suprathreshold effects can be explained by these shifts.

在海马 CA1 锥体神经元的树突中，阈下密度不均匀、快速失活的钾通道调节信号传播。 这种不均匀的分布（树突中的表达高于胞体中的表达）意味着树突的电特性与胞体的电特性明显不同。 输入的突触信号由这些通道的活动决定，而动作电位一旦在轴突中启动，随着它们传播回树突，其幅度逐渐减小。 分子神经生理学和生物物理学单元将膜片钳记录与分子技术相结合，研究 CA1 树突中表达的电压门控通道的电生理特性和分子性质、它们的表达如何受到调节以及它们在学习和记忆中的作用。 Kv4.2 对海马 CA1 锥体神经元放电模式的控制。 尽管最近的分子克隆研究发现了在哺乳动物大脑中表达的多个电压门控K通道基因家族，但目前缺乏有关这些基因的蛋白质产物与其各种神经元功能之间关系的信息。 我们的实验室结合了分子、电生理学、成像技术，证明电压门控钾通道亚基 Kv4.2 控制 AP 半宽度、频率依赖性 AP 展宽和树突动作电位传播。 由于 Ca2 流入主要发生在 AP 复极化期间，Kv4.2 活性可以调节涉及 Ca2 依赖性第二信使级联的细胞过程，例如基因表达和突触可塑性。 我们目前正在开发技术，使我们能够直接从树突记录我们已经改变的电压门控通道功能表达。 Kv4.2 在 CA1 锥体神经元树突中的运输。 使用改良的 Sinbis 病毒系统在培养的海马神经元中过表达 EGFP 标记的 Kv4.2 (Kv4.2g)，我们发现表达 Kv4.2g 的神经元的树突棘中的 EGFP 荧光看起来比相邻树突轴的 EGFP 荧光更亮。 表达 Kv4.2g 的神经元中棘头与树突轴荧光的比率大约是表达 EGFP 的神经元中的两倍。 与 NIH 的 Ron Petralia 合作，使用电子显微镜进一步显示了脊柱中的 Kv4.2 表达。 我们发现刺激 (AMPA) 会导致 Kv4.2g 从棘到树突轴的活性依赖性重新分布，以及 Kv4.2g 在胞体内的点状积累。 AMPA 诱导的 Kv4.2g 重新分布发生在刺激后 15 分钟内，并且是可逆的，表明该治疗不具有兴奋毒性（6 小时清除）。 与突触前和突触后标记（突触素和 NR1）的共表达表明，Kv4.2 经历了活动诱导的重新分布，而突触结构或数量没有发生总体变化。 我们通过响应 AMPA 刺激从脊柱移除 Kv4.2g 的实时成像证实了这些发现。 在这些条件下刺激的大量突触使我们能够直接测量内化的影响，即来自未感染的海马神经元的内源性全细胞瞬态 K 电流的减少，而不改变持续或非失活的延迟整流器型电压门控K 电流幅值。 因此，活性依赖性 Kv4.2 内化是天然发生的，而不是过度表达的产物。 我们目前正在调查 Kv4.2 活动依赖型贩运的要求和机制。 电压门控钾通道在突触可塑性中的作用。 使用 Sindbis 病毒系统用 Kv4.2g 和 Kv4.2g(W362F) 感染器官切片培养物，我们已开始使用去极化配对方案研究 Kv4.2 在 LTP 中的作用。 配对后的前 10 分钟，所有三组的增强效果相似，EPSC 大小增加了 100%。 然而，在此期间之后，Kv4.2 过度表达的神经元无法维持增强作用，使得 EPSC 大小在 25 分钟后回到基线。 相反，Kv4.2g(W362F) 的表达会导致增强，与对照相比，在启动后 40-50 分钟达到更高的水平。 这些数据表明，Kv4.2 通道通过影响增强后期的诱导或通过控制 LTP 维持机制来调节 LTP 程度。我们目前正在描述 Kv4.2s 对 LTP 影响的机制。 Kv4.2 转基因小鼠的创建和表征。 我们目前正在鉴定一种在电压门控钾通道亚基 Kv4.2 中表达显性负孔突变的转基因小鼠。 该小鼠在四环素反式激活因子 (tTA) 响应启动子的控制下表达突变型 Kv4.2 通道以及 GFP。 表达由表达 tTA 的新小鼠系在空间上控制，该小鼠将 tTA 活性限制在海马的 CA1 和齿状回区域。 可以通过施用多西环素暂时控制表达。 这些小鼠的急性海马切片实验将用于研究 Kv4.2 在调节 AP 反向传播到 CA1 树突以及突触整合和可塑性中的作用。 我们与 Anne Anderson 博士合作，正在研究这些小鼠的癫痫易感性。 此外，我们正在使用这些小鼠来研究 Kv4.2 在 Morris 水迷宫中海马依赖性学习和记忆中的作用。 辅助蛋白在调节 Kv4.2 表达和功能中的作用。 A 型 K 电流具有独特的动力学和电压依赖性特性，使它们能够微调突触事件、动作电位和神经元放电。 为了实现这种多样性，不同的神经元类型表达 Kv4.2 辅助亚基的特定互补体。 在海马 CA1 锥体神经元中，DPPX 是一个显着表达的亚基，当在异源系统中与 Kv4.2 共表达时，它可以恢复天然 CA1 A 型电流的许多特性。 为了研究 DPPX 在 CA1 神经元中的生理作用，我们与 Bernardo Rudys 实验室合作开发了短干扰 RNA (siRNA)，以抑制所有 DPPX 变体的表达。 为了研究 DPPX 是否改变本地系统中 A 型电流的动力学，我们使用改良的 Sindbis 病毒系统，对感染 siDPPX 的海马器官切片中的 CA1 锥体神经元的外侧斑块进行了电压钳实验。 我们发现 siDPPX 导致失活恢复延迟，并使 A 型电流的稳态失活和激活曲线右移。 为了确定 siDPPX A 型电流动力学修饰的生理效应，我们在 siDPPX 表达细胞中进行了电流钳实验。 与阴性对照相比，siDPPX 感染的神经元表现出输入阻力降低、AP 起始时间延迟、AP 阈值增加、AP 半宽度增加和快速 AHP 幅度降低。 因此，siDPPX 具有相反的效果，降低兴奋性阈下和增加兴奋性阈上。 我们使用计算机建模来确定这些阈下效应和阈上效应中的哪些可以通过这些变化来解释。